1. Field of Invention
The present invention relates to a novel mutant enzyme which synthesizes linear prenyl diphosphates that are precursors of compounds, important for organisms, such as steroids, ubiquinones, dolichols, carotenoids, prenylated proteins, animal hormones, plant hormones, and the like; a genetic system encoding said enzyme; and a method for producing and using said enzyme.
2. Related Art
Of the substances having important functions in organisms, many are biosynthesized using isoprene (2-methyl-1,3-butadiene) as a constituent units. These compounds are also called isoprenoids, terpenoids, or terpenes, and are classified depending on the number of carbon atoms into hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40), and the like. The actual biosynthesis starts with the mevalonate pathway through which mevalonic acid-5-diphosphate is synthesized, followed by the synthesis of isopentenyl diphosphate (IPP) which is an active isoprene unit.
The identity of the isoprene unit that was proposed as a precursor was found to be isopentenyl diphosphate, the so-called active isoprene unit. Dimethylallyl diphosphate (DMAPP), an isomer of isopentenyl diphosphate, being used as a substrate in the synthesis of isopentenyl adenine which is known as a cytokinin, one of the plant hormones, it is also known to undergo a condensation reaction with isopentenyl diphosphate to synthesize chain-form active isoprenoids such as geranyl diphosphate (GPP), neryl diphosphate, farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP), hexaprenyl diphosphate (HexPP), heptaprenyl diphosphate (HepPP), and the like.
There are Z type and E type condensation reactions. Geranyl diphosphate is a product of E type condensation and neryl diphosphate is of Z type condensation. Although, the all-E type is considered to be the active form in farnesyl diphosphate and geranylgeranyl diphosphate, the Z type condensation reaction leads to the synthesis of natural rubber, dolichols, bactoprenols (undecaprenols), and plants various polyprenols found in. They are believed to undergo the condensation reaction using the phosphate ester bond energy of the pyrophosphate and the carbon backbone present in the molecule and to produce pyrophosphate as the byproduct of the reaction.
Farnesyl diphosphate or geranylgeranyl diphosphate serve as a reaction substrate leading to the synthesis of prenylated proteins (from farnesyl diphosphate or geranylgeranyl diphosphate) represented by G proteins that are important in the mechanism of signal transducer in the cell; cell membrane lipids (from geranylgeranyl diphosphate) of archaea; squalene (from farnesyl diphosphate) which is a precursor of steroids; and phytoene (from geranylgeranyl diphosphate) which is a precursor of carotenoids. Prenyl diphosphates from hexaprenyl diphosphate and heptaprenyl diphosphate having six and seven isoprene units, respectively, to prenyl diphosphates having ten isoprene units serve as the precursors of the synthesis of ubiquinone and menaquinone (vitamin K2) that work in the electron transport system.
Furthermore, via the biosynthesis of these active-form isoprenoids, a vast number of kinds of compounds that are vital to life have been synthesized. Just to mention a few, there are cytokinins that are plant hormones and isopentenyl adenosine-modified tRNA that use hemiterpenes as their precursor of synthesis, geraniols and that isomer nerol belonging to monoterpens are the main components of rose oil perfume and a camphor tree extract, camphor, which is an insecticide. Sesquihormones include juvenile hormones of insects, diterpenes include a plant hormone gibberellin, trail pheromones of insects, and retinols and retinals that function as the visual pigment precursors, binding components of the purple membrane proteins of highly halophilic archaea, and vitamin A.
Furthermore, using squalene, a triterpene, a wide variety of steroid compounds have been synthesized, including, for example, animal sex hormones, vitamin D, ecdysone which is an ecdysis hormone of insects, a plant hormone brassinolide, constitution of the plasma membrane etc. Various carotenoids of tetraterpenes that are precursors of various pigments of organisms and vitamin A are also important compounds derived from active isoprenoids. Compounds such as chlorophyll, pheophytin, tocopherol (vitamin E), and phylloquinone (vitamin K1) are also derived from tetraterpenes.
The active isoprenoid synthases that sequentially condense isopentenyl diphosphates with such allylic substrates as dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, geranylfarnesyl diphosphate, etc. are called the prenyl diphosphate synthases, and are also called, based on the name of the compound having the maximum chain length of the major reaction products, for example farnesyl diphosphate synthase (FPP synthase), geranylgeranyl diphosphate (GGPP synthase), and the like. There are reports on purification, activity measurement, genetic cloning, and sequencing of the DNA encoding enzymes such as farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase, heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase, nonaprenyl diphosphate synthase (solanesyl diphosphate synthase), undecaprenyl diphosphate synthase, and the like from bacteria, archaea, fungi, plants, and animals.
These active isoprenoid synthases constituting the basis of chemical synthesis of a great variety of compounds that are important both in the industry and in the academic field of life sciences have had few practical uses in the industrial application due to their unstable nature and low specific activities. However, with the isolation of thermostable prenyl diphosphate synthases from thermophilic bacteria and archaea and the genes encoding these enzymes, their availability as the enzyme has increased.
With regard to farnesyl diphosphate synthase, a gene was isolated from Bacillus stearothermophilus, a medium thermophile, and an enzyme having a medium thermal stability was prepared using Escherichia coli as host cell T. Koyama et al. (1993) J. Biochem., 113: 355-363; Japanese Unexamined Patent Publication No. 5(1993)-219961!. With regard to geranylgeranyl diphosphate synthase, a gene was isolated from high thermophiles such as Sulfolobus acidocaldarius and Thermus thermophilus S. -i. Ohnuma et al., (1994) J. Biol. Chem., 269: 14792-14797; Japanese Unexamined Patent Publication No. 7(1995)-308193, and; Japanese Unexamined Patent Publication No. 7(1995)-294956!, and enzymes having a high thermal stability were prepared.
Furthermore, with regard to the prenyl diphosphate synthase having the functions of both of the farnesyl diphosphate synthase and the geranylgeranyl diphosphate synthase, the enzyme and the gene encoding it have been isolated from highly thermophile Methanobacterium thermoautotrophicum A. Chen and D. Poulter (1993) J. Biol. Chem., 268: 11002-11007; A. Chen and D. Poulter (1994) ARCHIVES OF BIOCHEMSTRY AND BIOPHYTSICS 314!, and the thermostable nature of the enzyme has been demonstrated.
However, in the synthesis of farnesyl diphosphate/geranylgeranyl diphosphate derived from Methanobacterium thermoautotrophicum, there are no reports on the data of thin layer chromatography analysis etc. that can specify the chain length of the reaction products in connection with the assay of the enzymatic activity; the chain length has been estimated by measuring geranyl diphosphate as the allylic substrate. Since geranyl diphosphate can also serve as a substrate of geranylgeranyl diphosphate synthase, it is unlikely that the measured activity includes that of the farnesyl diphosphate synthase alone.
Moreover, the presence of farnesyl diphosphate synthase has not been confirmed in archaea that are expected to have enzymes having higher thermo stability, higher salt-stability and lower-pH-stability.
As mentioned above, the use of the farnesyl diphosphate synthase derived from Bacillus stearothermophilus resolved part of the problem of the enzyme being unstable and difficult to handle. But, an enzyme having a higher thermal stability would be more stable and more amenable to industrial application.
Moreover, some prenyl diphosphate synthases having a longer chain length use farnesyl diphosphate as a substrate. When such a long-chain prenyl diphosphate synthase is used simultaneously with a farnesyl diphosphate synthase for the purpose of providing the substrate of the former enzyme, the latter enzyme must have stability which is equal to or higher than that of the long-chain prenyl diphosphate synthase. When industrial production of farnesyl diphosphate is contemplated, the enzyme must be immobilized or recovered for recycling. When it is regenerated, the enzyme itself to be more stable, must have higher thermo stability, higher salt stability, and higher stability in a wider range of pH.
It has been found out that of the two aspartic acid-rich domains that have been proposed based on the amino acid sequence of the prenyl diphosphate synthase, the amino acid residue located at the fifth position in the N-terminal direction from the conserved sequence I (DDXX(XX)D) (wherein X denotes any amino acid, and the two X's in the parentheses may not be present) of the aspartic acid-rich domain in the amino-terminal side is responsible for controlling the chain length of the reaction product. Hence, a method has been invented that controls the reaction product for the purpose of lengthening the chain length of the reaction product Japanese patent application No. 8-191635 filed on Jul. 3, 1996 under the title of "A Mutant Prenyl Diphosphate Synthase"!. The enzyme produced using the method enables production of reaction products that have several chain lengths. However, methods have not been not known that induce mutation of geranylgeranyl diphosphate synthase to control the reaction products to be in the short chain-length side in order to produce farnesyl diphosphate.